In a continuing endeavor to improve immunization against cholera, a search for a superior method of production of cholera toxoids will be conducted. Particular attention will be given to techniques adaptable to large scale, commercial production. The potential toxoids, resulting from chemical or enzymatic treatment, will be analyzed for antigenicity, immunogenicity in animals, residual toxicity, tissue receptor binding activity, and molecular structure. All resulting toxoids will be compared to the parent toxin and the currently available formaldehyde and glutaraldehyde toxoids. In order to continue our study of the mechanisms of intestinal immunity and to assist in quantitation of antigenicity of toxoids as indicated above a sensitive radioimmunoassay for cholera toxin will be developed. Similarly, a modification of the assay will be made to permit the quantitation of minute levels of cholera antitoxin, hopefully, with the capacity to distinguish between IgG, IgM, and IgA classes of antitoxin. Such an assay will be used to follow the antitoxin levels in serum, duodenal fluids, and feces from volunteers immunized parenterally with glutaraldehyde toxoid. If an oral toxoid were to be developed later, the procedure would be invaluable as a measure of systemic and intestinal antitoxin levels. Research and development on a parallel assay to quantitate antisomatic antibodies will be continued. A study of similar antibody levels in cholera patients will be conducted. It is also proposed to continue our research on cholera toxin binding to its tissue receptor. The presence of the receptor on duodenal mucosa will be determined. Attempts will be made in rabbits to study the protective role of antitoxin in blocking this binding phenomenon. Finally, it is further proposed to investigate the immunizing properties of a "combined vaccine" composed of purified Inaba antigen or whole cell vaccine and cholera toxoid in experimental animals and humans. The feasibility of using a "fringe" of active cholera toxin in this "combined vaccine" will also be studied in animals.